The long-term objective is to understand the mechanisms underlying diversification of the VH region in the human immunoglobulin heavy chain gene complex and the effect of the diversification on human health. The working hypothesis is that the differences resulted from the diversification in this region are of significant biological and genetic implications, and may contain "footprints" left by the genetic events responsible for the diversification. Study of these footprints may help to understand the mechanisms responsible for the VH region diversification. The VH region is highly diversified. It is believed that no haplotypes contain the same set of VH gene segments. Therefore, even if a complete nucleotide sequence for the VH region is generated by using diploid materials, the sequence may not represent the actual sequences in any haplotypes and is far from sufficient for understanding physical structure of the VH region. In the proposed study, 50 male human subjects with highly diversified genetic backgrounds will be recruited. VH gene segment number and composition in the donors' haplotypes will be determined by analyzing single haploid sperm cells from these donors. Four representative donors will be selected based on the results. The haplotype organization of the VH gene segments in these four subjects will be determined by analyzing single DNA fragments from single sperm. The resulting information will be used to organize the VH gene segment-containing clones from phage genomic libraries for these donors. The sequences of all VH gene segments and their flanking regions containing promoters, transcription regulatory elements, and recombination signal sequences in each haplotype will be determined. The detailed physical structure of each haplotype will be examined to identify the regions of genetic interest after restriction mapping. The sequences of the identified regions will be determined. The resulting data will be used to deduce the VH gene organization in the haplotypes of the remaining 46 individuals and for further analysis impact of the VH region diversification and the mechanisms underlying the diversification. The approaches developed in the study may also be used to analyze haplotype gene organization of other multi-gene families including those involved in the development of the immune system. And to determine the detailed physical structure of the chromosomal regions with single copy sequences.